Bio-barcodes based on oligonucleotide-modified particles

ABSTRACT

The present invention relates to a screening methods, compositions, and kits for detecting for the presence or absence of one or more target analytes, e.g. proteins such as antibodies, in a sample. In particular, the present invention relates to a method that utilizes reporter oligonucleotides as biochemical barcodes for detecting multiple protein structures or other target analytes in one solution.

CROSS-REFERENCE

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/820,279, filed Mar. 28, 2001 and claims the benefit of U.S.Provisional application Nos. 60/192,699, filed Mar. 28, 2000; and60/350,560, filed Nov. 13, 2001, which are incorporated by reference intheir entirety. The work reported in this application is funded, inpart, by NSF, ARO, and NIH grants. Accordingly, the U.S. government hascertain rights to the invention described in this application.

FIELD OF THE INVENTION

The present invention relates to a screening method for detecting forthe presence or absence of one or more proteins, e.g., antibodies, in asample. In particular, the present invention relates to a method thatutilizes reporter oligonucleotides as biochemical barcodes for detectingmultiple protein structures in one solution.

BACKGROUND OF THE INVENTION

The detection of proteins is important for both molecular biologyresearch and medical applications. Diagnostic methods based onfluorescence, mass spectroscopy, gell electrophoresis, laser scanningand electrochemistry are now available for identifying a variety ofprotein structures. ¹⁻⁴ Antibody-based reactions are widely used toidentify the genetic protein variants of blood cells, diagnose diseases,localize molecular probes in tissue, and purify molecules or effectseparation processes.⁵ For medical diagnostic applications (e.g. malariaand HIV), antibody tests such as the enzyme-linked immunosorbent assay,Western blotting, and indirect fluorescent antibody tests are extremelyuseful for identifying single target protein structures. ^(6,7) Rapidand simultaneous sample screening for the presence of multipleantibodies would be beneficial in both research and clinicalapplications. However, it is difficult, expensive, and time-consuming tosimultaneously detect several protein structures in one solution underhomogeneous assay conditions using the aforementioned related protocols.

SUMMARY OF THE INVENTION

The present invention relates to methods, compositions, and kits thatutilizes oligonucleotides as biochemical barcodes for detecting multipleprotein structures in one solution. The approach takes advantage ofprotein recognition elements functionalized with oligonucleotide strandsand the previous observation that hybridization events that result inthe aggregation of gold nanoparticles can significantly alter theirphysical properties (e.g. optical, electrical, mechanical).⁸⁻¹² Thegeneral idea is that each protein recognition element can be encodedwith a different oligonucleotide sequence with discrete and tailorablehybridization and melting properties and a physical signature associatedwith the nanoparticles that changes upon melting to decode a series ofanalytes in a multi-analyte assay.

In one embodiment of the invention, a method is provided for detectingfor the presence of a target analyte in a sample comprising:

-   -   providing a particle complex probe comprising a particle having        oligonucleotides bound thereto, a DNA barcode, and a        oligonucleotide having bound thereto a specific binding        complement to a target analyte, wherein the DNA barcode has a        sequence having at least two portions, at least some of the        oligonucleotides attached to the particle have a sequence that        is complementary to a first portion of a DNA barcode, the        oligonucleotides having bound thereto a specific binding        complement have a sequence that is complementary to a second        portion of a DNA barcode, and wherein the DNA barcode is        hybridized at least to some of the oligonucleotides attached to        the particle and to the oligonucleotides having bound thereto        the specific binding complement;    -   contacting the sample with a particle complex probe under        conditions effective to allow specific binding interactions        between the analyte and the particle complex probe and to form        an aggregated complex in the presence of analyte; and    -   observing whether aggregate formation occurred.

In the presence of target analyte, aggregates are produced as a resultof the binding interactions between the particle complex probe and thetarget analyte. The aggregates may be detected by any suitable means.

In another embodiment of the invention, a method is provided fordetecting for the presence of one or more target analytes in a samplecomprising:

-   -   providing one or more types of particle complex probe comprising        a particle having oligonucleotides bound thereto, a DNA barcode,        and a oligonucleotide having bound thereto a specific binding        complement to a specific target analyte, wherein (i) the DNA        barcode has a sequence having at least two portions, (ii) at        least some of the oligonucleotides attached to the particle have        a sequence that is complementary to a first portion of a DNA        barcode, (iii) the oligonucleotides having bound thereto a        specific binding complement have a sequence that is        complementary to a second portion of a DNA barcode, and (iv) the        DNA barcode in each type of particle complex probe has a        sequence that is different and that serves as an identifier for        a particular target analyte;    -   contacting the sample with a particle complex probe under        conditions effective to allow specific binding interactions        between the analyte and the particle complex probe and to form        an aggregated complex in the presence of analyte;    -   isolating aggregated complexes; and    -   analyzing the aggregated complexes to determine the presence of        one or more DNA barcodes having different sequences.

Each type of particle complex probe contains a predetermined reporteroligonucleotde or barcode for a particular target analyte. In thepresence of target analyte, nanoparticle aggregates are produced as aresult of the binding interactions between the nanoparticle complex andthe target analyte. These aggregates can be isolated and analyzed by anysuitable means, e.g., thermal denaturation, to detect the presence ofone or more different types of reporter oligonucleotides.

In yet another embodiment of the invention, a method is provided fordetecting for the presence of a target analyte in a sample comprising:

-   -   providing a particle complex probe comprising a particle having        oligonucleotides bound thereto, a DNA barcode, and a        oligonucleotide having bound thereto a specific binding        complement to a target analyte, wherein the DNA barcode has a        sequence having at least two portions, at least some of the        oligonucleotides attached to the particle have a sequence that        is complementary to a first portion of a DNA barcode, the        oligonucleotides having bound thereto a specific binding        complement have a sequence that is complementary to a second        portion of a DNA barcode, and wherein the DNA barcode is        hybridized at least to some of the oligonucleotides attached to        the particle and to the oligonucleotides having bound thereto        the specific binding complement;    -   contacting the sample with a particle complex probe under        conditions effective to allow specific binding interactions        between the analyte and the particle complex probe and to form        an aggregated complex in the presence of analyte;    -   isolating the aggregated complex and subjecting the aggregated        complex to conditions effective to dehybridize the aggregated        complex and to release the DNA barcode;    -   isolating the DNA barcode; and    -   detecting for the presence of DNA barcode.

In the presence of target analyte, nanoparticle aggregates are producedas a result of the binding interactions between the nanoparticle complexand the target analyte. These aggregates are isolated and subject toconditions effective to dehybridize the aggregate and to release thereporter oligonucleotide. The reporter oligonucleotide is then isolated.If desired, the reporter oligonucleotide may be amplified by anysuitable means including PCR amplification. Analyte detection occursindirectly by ascertaining for the presence of reporter oligonucleotideor biobarcode by any suitable means such as a DNA chip.

In yet another embodiment of the invention, a method for detecting forthe presence of one or more target analytes in a sample comprising:

-   -   providing one or more types of particle complex probe comprising        a particle having oligonucleotides bound thereto, a DNA barcode,        and a oligonucleotide having bound thereto a specific binding        complement to a specific target analyte, wherein (i) the DNA        barcode has a sequence having at least two portions, (ii) at        least some of the oligonucleotides attached to the particle have        a sequence that is complementary to a first portion of a DNA        barcode, (iii) the oligonucleotides having bound thereto a        specific binding complement have a sequence that is        complementary to a second portion of a DNA barcode, and (iv) the        DNA barcode in each type of particle complex probe has a        sequence that is different and that serves as an identifier for        a particular target analyte;    -   contacting the sample with a particle complex probe under        conditions effective to allow specific binding interactions        between the analyte and the particle complex probe and to form        aggregated complexes in the presence of one or more analytes;    -   isolating the aggregated complexes and subjecting the aggregated        complexes to conditions effective to dehybridize the aggregated        complexes and to release the DNA barcodes;    -   isolating the DNA barcodes; and    -   detecting for the presence of one or more DNA barcodes having        different sequences, wherein the identification of a particular        DNA barcode is indicative of the presence of a specific target        analyte in the sample.

In the presence of one or more target analyte, aggregates are producedas a result of the binding interactions between the particle complexprobe and the target analyte. These aggregates are isolated and subjectto conditions effective to dehybridize the aggregate and to release thereporter oligonucleotides. The reporter oligonucleotides is thenisolated. If desired, the reporter oligonucleotide may be amplified byany suitable means including PCR amplification. Analyte detection occursindirectly by ascertaining for the presence of reporter oligonucleotideor biobarcode by any suitable means such as a DNA chip.

In yet another embodiment of the invention, a method for detecting forthe presence of one or more antibodies in a sample comprising:

-   -   providing one or more types of particle complex probe comprising        a particle having oligonucleotides bound thereto, a DNA barcode,        and a oligonucleotide having bound thereto a hapten to a        specific target antibody, wherein (i) the DNA barcode has a        sequence having at least two portions, (ii) at least some of the        oligonucleotides attached to the particle have a sequence that        is complementary to a first portion of a DNA barcode, (iii) the        oligonucleotides having bound thereto a hapten to a specific        target antibody have a sequence that is complementary to a        second portion of a DNA barcode, and (iv) the DNA barcode in        each type of particle complex probe has a sequence that is        different and that serves as an identifier for a particular        target antibody;    -   contacting the sample with a particle complex probe under        conditions effective to allow specific binding interactions        between the antibody and the particle complex probe and to form        aggregated complexes in the presence of one or more target        antibodies;    -   isolating the aggregated complexes and subjecting the aggregated        complexes to conditions effective to dehybridize the aggregated        complexes and to release the DNA barcodes;    -   isolating the DNA barcodes; and    -   detecting for the presence of one or more DNA barcodes having        different sequences, wherein the identification of a particular        DNA barcode is indicative of the presence of a specific target        antibody.

The invention also provides a method is provided for detecting for thepresence of a target analyte in a sample which entails generating aparticle complex probe in situ. In one embodiment of the invention, amethod for detecting for the presence of target analytes comprises:

-   -   providing particles having oligonucleotides bound thereto, DNA        barcodes, and oligonucleotides having bound thereto a specific        binding complement to a target analyte, wherein the DNA barcodes        have a sequence with at least two portions, at least some of the        oligonucleotides attached to the particles have a sequence that        is complementary to a first portion of a DNA barcode, the        oligonucleotides having bound thereto a specific binding        complement have a sequence that is complementary to a second        portion of a DNA barcodes    -   contacting the sample with a particle complex probe under        conditions effective to allow hybridization of the DNA barcode        to at least to some of the oligonucleotides attached to the        particle and to the oligonucleotides having bound thereto the        specific binding complement and to allow specific binding        interactions between the analyte and the oligonucleotides having        bound thereto a specific binding complement to the analyte, said        contacting resulting in the formation of an aggregated complex        in the presence of analyte; and    -   observing whether aggregate formation occurred.

In another embodiment of the invention, a method is provided fordetecting for the presence of one or more target analytes in a samplecomprising:

-   -   providing one or more types of particles having oligonucleotides        bound thereto, one or more types of DNA barcodes, and one or        more types of oligonucleotides having bound thereto a specific        binding complement to a specific target analyte, wherein (i)        each type of DNA barcode has a sequence having at least two        portions, (ii) at least some of the oligonucleotides attached to        the particle have a sequence that is complementary to a first        portion of one or more types of DNA barcode, (iii) each type of        oligonucleotide having bound thereto a specific binding        complement have a sequence that is complementary to a second        portion of a type of DNA barcode, and (iv) each type of DNA        barcode serves as an identifier for a particular target analyte        and has a sequence that is different from another type of DNA        barcode;    -   contacting the sample with one or more types of particles having        oligonucleotides bound thereto, one or more types of DNA        barcodes, and one or more types of oligonucleotides having bound        thereto a specific binding complement to a specific target        analyte, under conditions effective to allow hybridization of        each type of DNA barcodes at least to some of the        oligonucleotides attached to the particles and to a type of        oligonucleotides having bound thereto the specific binding        complement and to allow specific binding interactions between a        specific target analyte and a type of oligonucleotides having        bound thereto a specific binding complement to the specific        target analyte, said contacting resulting in the formation of        aggregated complexes in the presence of one or more specific        target analytes;    -   isolating aggregated complexes; and    -   analyzing the aggregated complexes to determine the presence of        one or more DNA barcode, where the presence of a particular DNA        barcode is indicative of the presence of a specific target        analyte in the sample.

In yet another embodiment of the invention, a method is provided fordetecting for the presence of a target analyte in a sample comprising:

-   -   providing particles having oligonucleotides bound thereto, DNA        barcodes, and oligonucleotides having bound thereto a specific        binding complement to a target analyte, wherein the DNA barcodes        have a sequence with at least two portions, at least some of the        oligonucleotides attached to the particles have a sequence that        is complementary to a first portion of a DNA barcode, the        oligonucleotides having bound thereto a specific binding        complement have a sequence that is complementary to a second        portion of a DNA barcodes;    -   contacting the sample with a particle complex probe under        conditions effective to allow hybridization of the DNA barcode        to at least to some of the oligonucleotides attached to the        particle and to the oligonucleotides having bound thereto the        specific binding complement and to allow specific binding        interactions between the analyte and the oligonucleotides having        bound thereto a specific binding complement to the analyte, said        contacting resulting in the formation of an aggregated complex        in the presence of analyte;    -   isolating the aggregated complex and subjecting the aggregated        complex to conditions effective to dehybridize the aggregated        complex and to release the DNA barcode;    -   isolating the DNA barcode; and    -   detecting for the presence of DNA barcode.

In yet another embodiment of the invention, a method is provided fordetecting for the presence of one or more target analytes in a samplecomprising:

-   -   providing one or more types of particles having oligonucleotides        bound thereto, one or more types of DNA barcodes, and one or        more types of oligonucleotides having bound thereto a specific        binding complement to a specific target analyte, wherein (i)        each type of DNA barcode has a sequence having at least two        portions, (ii) at least some of the oligonucleotides attached to        the particle have a sequence that is complementary to a first        portion of one or more types of DNA barcode, (iii) each type of        oligonucleotide having bound thereto a specific binding        complement have a sequence that is complementary to a second        portion of a type of DNA barcode, and (iv) each type of DNA        barcode serves as an identifier for a particular target analyte        and has a sequence that is different from another type of DNA        barcode;    -   contacting the sample with one or more types of particles having        oligonucleotides bound thereto, one or more types of DNA        barcodes, and one or more types of oligonucleotides having bound        thereto a specific binding complement to a specific target        analyte, under conditions effective to allow hybridization of        each type of DNA barcodes at least to some of the        oligonucleotides attached to the particles and to a type of        oligonucleotides having bound thereto the specific binding        complement and to allow specific binding interactions between a        specific target analyte and a type of oligonucleotides having        bound thereto a specific binding complement to the specific        target analyte, said contacting resulting in the formation of        aggregated complexes in the presence of one or more specific        target analytes;    -   isolating the aggregated complexes and subjecting the aggregated        complexes to conditions effective to dehybridize the aggregated        complexes and to release the DNA barcodes;    -   isolating the DNA barcodes; and    -   detecting for the presence of one or more DNA barcodes having        different sequences, wherein the identification of a particular        DNA barcode is indicative of the presence of a specific target        analyte.

In yet another embodiment of the invention, a method is provided fordetecting for the presence of one or more antibodies in a samplecomprising:

-   -   providing one or more types of particles having oligonucleotides        bound thereto, one or more types of DNA barcodes, and one or        more types of oligonucleotides having bound thereto a hapten to        a specific antibody, wherein (i) each type of DNA barcode has a        sequence having at least two portions, (ii) at least some of the        oligonucleotides attached to the particle have a sequence that        is complementary to a first portion of one or more types of DNA        barcode, (iii) each type of oligonucleotide having bound thereto        a hapten to a specific antibody has a sequence that is        complementary to a second portion of a type of DNA barcode,        and (iv) each type of DNA barcode serves as an identifier for a        particular target antibody and has a sequence that is different        from another type of DNA barcode;    -   contacting the sample with one or more types of particles having        oligonucleotides bound thereto, one or more types of DNA        barcodes, and one or more types of oligonucleotides having bound        thereto a hapten to a specific target antibody, under conditions        effective to allow hybridization of each type of DNA barcodes at        least to some of the oligonucleotides attached to the particles        and to a type of oligonucleotides having bound thereto the        hapten and to allow specific binding interactions between a        specific target antibody and a type of oligonucleotides having        bound thereto a hapten to the specific target antibody, said        contacting resulting in the formation of aggregated complexes in        the presence of one or more specific target antibodies;    -   isolating the aggregated complexes and subjecting the aggregated        complexes to conditions effective to dehybridize the aggregated        complexes and to release the DNA barcodes;    -   isolating the DNA barcodes; and    -   detecting for the presence of one or more DNA barcodes having        different sequences, wherein the identification of a particular        DNA barcode is indicative of the presence of a specific target        antibody.

The invention also provides kits for target analyte detection. In oneembodiment of the invention, a kit is provided for detecting a targetanalyte in a sample, the kit comprising at least one container includingparticle complex probes comprising a particle having oligonucleotidesbound thereto, a DNA barcode, and a oligonucleotide having bound theretoa specific binding complement to a target analyte, wherein the DNAbarcode has a sequence having at least two portions, at least some ofthe oligonucleotides attached to the particle have a sequence that iscomplementary to a first portion of a DNA barcode, the oligonucleotideshaving bound thereto a specific binding complement have a sequence thatis complementary to a second portion of a DNA barcode, and wherein theDNA barcode is hybridized to at least to some of the oligonucleotidesattached to the particle and to the oligonucleotides having boundthereto the specific binding complement, and an optional substrate forobserving a detectable change.

In another embodiment of the invention, a kit is provided for detectingone or more target analytes in a sample, the kit comprising at least oneor more containers, container holds a type of particle complex probecomprising a particle having oligonucleotides bound thereto, a DNAbarcode, and a oligonucleotide having bound thereto a specific bindingcomplement to a specific target analyte, wherein (i) the DNA barcode hasa sequence having at least two portions, (ii) at least some of theoligonucleotides attached to the particle have a sequence that iscomplementary to a first portion of a DNA barcode, (iii) theoligonucleotides having bound thereto a specific binding complement havea sequence that is complementary to a second portion of a DNA barcode,and (iv) the DNA barcode in each type of particle complex probe has asequence that is different and that serves as an identifier for aparticular target analyte; wherein the kit optionally includes asubstrate for observing a detectable change.

In yet another embodiment of the invention, a kit is provided for thedetection of a target analyte, the kit includes at least one pair ofcontainers and an optional substrate for observing a detectable change,

-   -   the first container of the pair includes particle probe        comprising a particle having oligonucleotides bound thereto and        a DNA barcode having a sequence of at least two portions,        wherein at least some of the oligonucleotides attached to the        particle have a sequence that is complementary to a first        portion of a DNA barcode;    -   the second container of the pair includes an oligonucleotide        having a sequence that is complementary to a second portion of        the DNA barcode, the oligonucleotide having a moiety that can be        used to covalently link a specific binding pair complement of a        target analyte.

In yet another embodiment of the invention, a kit is provided for thedetection of multiple target analytes in a sample, the kit includes atleast two or more pairs of containers,

-   -   the first container of each pair includes particle complex        probes having particles having oligonucleotides bound thereto        and a DNA barcode having a sequence of at least two portions,        wherein at least some of the oligonucleotides bound to the        particles have a sequence that is complementary to a first        portion of a DNA barcode having at least two portions; and    -   the second container of each pair contains a oligonucleotide        having a sequence that is complementary to a second portion of        the DNA barcode, the oligonucleotide having a moiety that can be        used to covalently link a specific binding pair complement of a        target analyte,    -   wherein the DNA barcode for type of particle complex probe has a        sequence that is different and that serves as an identifer for a        target analyte and wherein the kit optionally include a        substrate for observing a detectable change.

In yet another embodiment of the invention, a kit is provided for thedetection of multiple target analytes in a sample, the kit includes afirst container and at least two or more pairs of containers,

-   -   the first container includes particle complex probes having        particles having oligonucleotides bound thereto;    -   the first container of the pair includes a DNA barcode having a        sequence of at least two portions, wherein at least some of the        oligonucleotides bound to the particles have a sequence that is        complementary to a first portion of the DNA barcode; and    -   the second container of each pair contains a oligonucleotide        having a sequence that is complementary to a second portion of        the DNA barcode, the oligonucleotide having a moiety that can be        used to covalently link a specific binding pair complement of a        target analyte,    -   wherein the DNA barcode present in the first container of each        pair of containers serves as an identifer for a target analyte        and has a sequence that is different from a DNA barcode in        another pair of containers, and wherein the kite optionally        include a substrate for observing a detectable change.

In yet another embodiment of the invention, the particle of any of theforegoing kits may comprise a nanoparticle such as metal, semiconductor,insulator, or magnetic nanoparticles, preferably gold nanoparticles.

The invention also includes a system for detecting one or more targetanalytes in a sample comprising:

-   -   one or more types of particle complex probes, each particle        complex probe comprising a particle having oligonucleotides        bound thereto, a DNA barcode, and a oligonucleotide having bound        thereto a specific binding complement to a specific target        analyte, wherein (i) the DNA barcode has a sequence having at        least two portions, (ii) at least some of the oligonucleotides        attached to the particle have a sequence that is complementary        to a first portion of a DNA barcode, (iii) the oligonucleotide        having bound thereto a specific binding complement have a        sequence that is complementary to a second portion of a DNA        barcode, and (iv) the DNA barcode in each type of particle        complex probe has a sequence that is different and that serves        as an identifier for a particular target analyte.

The particle in the system preferably comprises a nanoparticle such asmetal, semiconductor, insulator, or magnetic nanoparticles, preferablygold nanoparticles.

The invention also comprises a particle complex probe comprising aparticle having oligonucleotides bound thereto, a DNA barcode, and aoligonucleotide having bound thereto a specific binding complement to atarget analyte, wherein the DNA barcode has a sequence having at leasttwo portions, at least some of the oligonucleotides attached to theparticle have a sequence that is complementary to a first portion of aDNA barcode, the oligonucleotides having bound thereto a specificbinding complement have a sequence that is complementary to a secondportion of a DNA barcode, and wherein the DNA barcode is hybridized atleast to some of the oligonucleotides attached to the particle and tothe oligonucleotides having bound thereto the specific bindingcomplement. The particle in the probe preferably comprises ananoparticle such as metal, semiconductor, insulator, or magneticnanoparticles, preferably gold nanoparticles.

The invention also includes an oligonucleotide sequence having boundthereto a specific target complement to a target analyte.

The invention also includes a DNA barcode comprising a oligonucleotidesequence that serves as an identifier for the presence of a specifictarget analyte.

The invention also includes two or more DNA barcodes comprising anoligonucleotide sequence, each DNA barcode having a differentoligonucleotide sequence and serving as an identifier for the presenceof a specific target analyte.

As used herein, a “type of” nanoparticles, conjugates, particles, latexmicrospheres, etc. having oligonucleotides attached thereto refers to aplurality of that item having the same type(s) of oligonucleotidesattached to them. “Nanoparticles having oligonucleotides attachedthereto” or “Nanoparticles having oligonucleotides attached thereto” arealso sometimes referred to as “nanoparticle-oligonucleotide conjugates”or, in the case of the detection methods of the invention,“nanoparticle-oligonucleotide probes,” “nanoparticle probes,” or just“probes.”

The term “nanoparticle complex” or “nanoparticle complex probe” refersto a conjugate comprised of nanoparticle-oligonucleotide conjugates, areporter oligonucleotide, and an oligonucleotide having bound thereto aspecific binding complement to a target analyte.

The term “analyte” refers to the compound or composition to be detected,including drugs, metabolites, pesticides, pollutants, and the like. Theanalyte can be comprised of a member of a specific binding pair (sbp)and may be a ligand, which is monovalent (monoepitopic) or polyvalent(polyepitopic), usually antigenic or haptenic, and is a single compoundor plurality of compounds which share at least one common epitopic ordeterminant site. The analyte can be a part of a cell such as bacteriaor a cell bearing a blood group antigen such as A, B, D, etc., or an HLAantigen or a microorganism, e.g., bacterium, fungus, protozoan, orvirus.

The polyvalent ligand analytes will normally be poly(amino acids), i.e.,polypeptides and proteins, polysaccharides, nucleic acids, andcombinations thereof. Such combinations include components of bacteria,viruses, chromosomes, genes, mitochondria, nuclei, cell membranes andthe like.

For the most part, the polyepitopic ligand analytes to which the subjectinvention can be applied will have a molecular weight of at least about5,000, more usually at least about 10,000. In the poly(amino acid)category, the poly(amino acids) of interest will generally be from about5,000 to 5,000,000 molecular weight, more usually from about 20,000 to1,000,000 molecular weight; among the hormones of interest, themolecular weights will usually range from about 5,000 to 60,000molecular weight.

A wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc. Such proteins include,for example, immunoglobulins, cytokines, enzymes, hormones, cancerantigens, nutritional markers, tissue specific antigens, etc.

The types of proteins, blood clotting factors, protein hormones,antigenic polysaccharides, microorganisms and other pathogens ofinterest in the present invention are specifically disclosed in U.S.Pat. No. 4,650,770, the disclosure of which is incorporated by referenceherein in its entirety.

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.

The analyte may be a molecule found directly in a sample such as a bodyfluid from a host. The sample can be examined directly or may bepretreated to render the analyte more readily detectible. Furthermore,the analyte of interest may be determined by detecting an agentprobative of the analyte of interest such as a specific binding pairmember complementary to the analyte of interest, whose presence will bedetected only when the analyte of interest is present in a sample. Thus,the agent probative of the analyte becomes the analyte that is detectedin an assay. The body fluid can be, for example, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, and the like.

The term “specific binding pair (sbp) member” refers to one of twodifferent molecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These will usually be members of an immunologicalpair such as antigen-antibody, although other specific binding pairssuch as biotin-avidin, hormones-hormone receptors, nucleic acidduplexes, IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA,and the like are not immunological pairs but are included in theinvention and the definition of sbp member.

The term “ligand” refers to any organic compound for which a receptornaturally exists or can be prepared. The term ligand also includesligand analogs, which are modified ligands, usually an organic radicalor analyte analog, usually of a molecular weight greater than 100, whichcan compete with the analogous ligand for a receptor, the modificationproviding means to join the ligand analog to another molecule. Theligand analog will usually differ from the ligand by more thanreplacement of a hydrogen with a bond which links the ligand analog to ahub or label, but need not. The ligand analog can bind to the receptorin a manner similar to the ligand. The analog could be, for example, anantibody directed against the idiotype of an antibody to the ligand.

The term “receptor” or “antiligand” refers to any compound orcomposition capable of recognizing a particular spatial and polarorganization of a molecule, e.g., epitopic or determinant site.Illustrative receptors include naturally occurring receptors, e.g.,thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins,nucleic acids, avidin, protein A, barstar, complement component C1q, andthe like. Avidin is intended to include egg white avidin and biotinbinding proteins from other sources, such as streptavidin.

The term “specific binding” refers to the specific recognition of one oftwo different molecules for the other compared to substantially lessrecognition of other molecules. Generally, the molecules have areas ontheir surfaces or in cavities giving rise to specific recognitionbetween the two molecules. Exemplary of specific binding areantibody-antigen interactions, enzyme-substrate interactions,polynucleotide interactions, and so forth.

The term “non-specific binding” refers to the non-covalent bindingbetween molecules that is relatively independent of specific surfacestructures. Non-specific binding may result from several factorsincluding hydrophobic interactions between molecules.

The term “antibody” refers to an immunoglobulin which specifically bindsto and is thereby defined as complementary with a particular spatial andpolar organization of another molecule. The antibody can be monoclonalor polyclonal and can be prepared by techniques that are well known inthe art such as immunization of a host and collection of sera(polyclonal) or by preparing continuous hybrid cell lines and collectingthe secreted protein (monoclonal), or by cloning and expressingnucleotide sequences or mutagenized versions thereof coding at least forthe amino acid sequences required for specific binding of naturalantibodies. Antibodies may include a complete immunoglobulin or fragmentthereof, which immunoglobulins include the various classes and isotypes,such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragmentsthereof may include Fab, Fv and F(ab′).sub.2, Fab′, and the like. Inaddition, aggregates, polymers, and conjugates of immunoglobulins ortheir fragments can be used where appropriate so long as bindingaffinity for a particular molecule is maintained.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a DNA/Au nanoparticle-based protein detection scheme.(A) Preparation of hapten-modified nanoparticle probes. (B) Proteindetection using protein binding probes. Notice that there are nine G,Cpairs in sequence A and there are only two G,C pairs in sequence B.

FIG. 2 illustrates thermal denaturation profiles for Au nanoparticleaggregates linked by DNA and proteins. Extinction at 260 nm wasmonitored as a function of increasing temperature (1° C./min, 1 minholding time). Each UV-Vis spectrum was measured under constant stirringto suspend the aggregates. All the aggregates were suspended in 1 ml of0.3 M PBS prior to performing the melting analyses. A) Two probes withone target antibody present IgE (

), IgG1 (---)); all data have been normalized; (B) Two probes with bothtarget antibodies present. Inset; first derivative of the thermaldenaturation curve.

FIG. 3 illustrates an array-based protein detection scheme using DNA asa biobarcode for the protein.

FIG. 4 illustrates scanometric DNA array detection of the DNAbiobarcodes. Left column is for the detection of the biobarcodeassociated with IgG1 and the right column is for the biobarcodeassociated with IgE. The capture oligonucleotides are 5′-thiol-modifiedATAACTAGAACTTGA for the IgG1 system and 5′-thiol-modifed TTATCTATTATTfor the IgE system. Each spot is approximately 250 um in diameter andread via gray-scale with an Epson Expression 1640XL flatbed scanner(Epson America, Longbeach, Calif.). These assays have been studied andwork comparably well over the 20 nM to 700 nM target concentrationrange.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method that utilizes oligonucleotidesas biochemical barcodes for detecting multiple protein structures in onesolution (FIG. 1). The approach takes advantage of protein recognitionelements functionalized with oligonucleotide strands and the previousobservation that hybridization events that result in the aggregation ofgold nanoparticles can significantly alter their physical properties(e.g. optical, electrical, mechanical).⁸⁻¹² The general idea is thateach protein recognition element can be encoded with a differentoligonucleotide sequence with discrete and tailorable hybridization andmelting properties and a physical signature associated with thenanoparticles that changes upon melting to decode a series of analytesin a multi-analyte assay. Therefore, one can use the melting temperatureof a DNA-linked aggregate and a physical property associated with thenanoparticles that changes upon melting to decode a series of analytesin a multi-analyte assay. The barcodes herein are different from theones based on physical diagnostic markers such as nanorods,²³flourophore-labeled beads,²⁴ and quantum dots,²⁵ in that the decodinginformation is in the form of chemical information stored in apredesigned oligonucleotide sequence.

In one aspect of the invention, a method for detecting for the presenceof a target analyte, e.g., an antibody, in a sample is provided. Anantibody such as immunoglobulin E (IgE) or immunoglobulin G1 (IgG1)shown in the Examples below can be detected with olignucleotide-modifiedprobes prehybridized with oligonucleotide strands modified with theappropriate hapten (biotin in the case of IgG1 and dinitrophenyl (DNP)in the case of IgE; FIG. 1A). ^(13,14) The DNA sequences in theproof-of-concept assays presented in the Examples below were designed ina way that would ensure that the two different aggregates formed fromthe probe reactions with IgG1 and IgE would melt at differenttemperatures, FIG. 1B. The probes for IgG1 have longer sequences andgreater G,C base contents than those for IgE. Therefore, the formersequences melt at a higher temperature than the latter ones. Thesesequence variations allow one to prepare probes with distinct meltingsignatures that can be used as codes to identify which targets havereacted with them to form nanoparticle aggregates. Three differentsystems have been studied: (1) two probes with one target antibodypresent (IgG1 or IgE); (2) two probes with the two different targetantibodies present, and (3) a control where no target antibodies arepresent.

In this aspect of the invention, a method is provided for detecting thepresence of a target analyte, e.g., an antibody, in a sample comprisescontacting a nanoparticle probe having oligonucleotides bound theretowith a sample which may contain a target analyte. At least some of theoligonucleotides attached to the nanoparticle are bound to a firstportion of a reporter oligonucleotide as a result of hybridization. Asecond portion of the reporter oligonucleotide is bound, as a result ofhybridization, to an oligonucleotide having bound thereto a specificbinding complement (e.g., antigen) to the analyte. The contacting takesplace under conditions effective to allow specific binding interactionsbetween the analyte and the nanoparticle probe. In the presence oftarget analyte, nanoparticle aggregates are produced. These aggregatesmay be detected by any suitable means.

In practicing the invention, a nanoparticle complex probes are preparedby hybridizing the nanoparticles having oligonucleotides bound theretowith an oligonucleotide modified with a specific binding complement to atarget analyte, and a reporter oligonucleotide. At least some of theoligonucleotides attached to the nanoparticle have a sequence that iscomplementary to a first portion of a reporter oligonucleotide. Theoligonucleotides having bound thereto a specific binding complement havea sequence that is complementary to a second portion of a reporteroligonucleotide. The reporter oligonucleotide hybridizes to the at leastto some of the oligonucleotides attached to the nanoparticle and to theoligonucleotides having bound thereto the specific binding complement,forming the nanoparticle complex probe under conditions sufficient toallow for hybridization between the components. Any suitable solventmedium and hybridization conditions may be employed in preparing thenanoparticle complex solution that allows for sufficient hybridizationof the components. Preferably, the components are hybridized in aphosphate buffered solution (PBS) comprised of 0.3 M NaCl and 10 mMphosphate buffer (pH 7) at room temperature for about 2-3 hours. Theconcentration of nanoparticle-oligonucleotide conjugates in thehybridization mixture range between about 2 and about 50, preferablyabout 13 nM. The concentration of hapten-modified oligonucleotidesgenerally ranges between about 50 and about 900, preferably about 300nM. The concentration of reporter oligonucleotide generally rangesbetween about 50 and about 900, preferably about 300 nM. Unreactedhapten-modified oligonucleotide and reporter oligonucleotides may beoptionally, but preferably, removed by any suitable means, preferablyvia centrifugation (12,000 rpm, 20 minutes) of the hybridization mixtureand subsequent decanting of the supernatant. The prepared complexes werestored in 0.3 M NaCl and 10 mM phosphate buffer (pH 7-7.4), 0.01% azidesolution at 4-6° C.

A typical assay for detecting the presence of a target analyte, e.g,antibody, in a sample is as follows: a solution containing nanoparticlecomplex probe comprising nanoparticles having oligonucleotides boundthereto, a reporter oligonucleotide, and an oligonucleotide having aspecific binding complement to the target analyte, is admixed with anaqueous sample solution believed to contain target protein. The totalprotein content in the aqueous sample solution generally ranges betweenabout 5 and about 100, usually about 43 ug/ml. The concentration ofnanoparticles in the reaction mixture generally ranges between about 2and about 20, usually about ˜13 nM. The total volume of the resultingmixture generally ranges between about 100 and about 1000, preferablyabout 400 uL. Any suitable solvent may be employed in preparing theaqueous sample solution believed to contain target analyte, preferablyPBS comprising 0.3 M NaCl and 10 mM phosphate buffer (pH 7-7.4).

The resulting assay mixture is then incubated at a temperature rangingbetween about 35 and about 40° C., preferably at 37° C., for a timeranging between about 30 and about 60, preferably about 50 minutes,sufficient to facilitate specific binding pair, e.g., protein-hapten,complexation. If the target protein is present, particle aggregationtakes place effecting a shift in the gold nanoparticle plasmon band anda red-to-purple color change along with precipitation. The hybridizedproducts are centrifuged (e.g., 3000 rpm for 2 minutes), and thesupernatant containing unreacted elements are decanted prior toanalysis.

If desired, the nanoparticle complex probe may be prepared in situwithin the assay mixture by admixing all the nanoparticles havingoligonucleotides bound thereto, the reporter oligonucleotide, and thehapten-modified oligonucleotide with the sample suspected of containinga target analyte. To ensure complete hybridization among all thecomponents, especially the complementary DNA strands, the assay mixturemay be incubated to expedite hybridization at −15° C. for 20 minutes(Boekel Tropicooler Hot/Cold Block Incubator) and stored at 4° C. for 24hours. In practicing the invention, however, it is preferred that thenanoparticle complex probe is prepared prior to conducting the assayreaction to increase the amount of DNA barcode within the nanoparticlecomplex probe.

To determine which proteins are present, a melting analysis of theaggregates which monitors the extinction at 260 nm as a function oftemperature may carried out in the solution. See, for instance, FIG. 2in Example 3 which describes analysis of a sample containing one or twoknown target analytes: IgG1 and IgE. As discussed in Example 3, whenIgG1 is treated with the probes via the aforementioned protocol, thesolution turns pinkish-blue, indicating the formation of nanoparticleaggregates. In a control experiment where no target but backgroundproteins are present, there is no discernible precipitation. A meltinganalysis of the solution shows a sharp transition with a meltingtemperature (Tm) of 55° C. This is the expected transition for the IgG1target, FIG. 2A (---). If IgE is added to a fresh solution of probes,the same color change is observed but the melting analysis provides acurve with a Tm of 36° C., the expected transition for this target, FIG.2A (

). Significantly, when both protein targets are added to the solution ofprobes, the solution turns dark purple, and the melting analysisexhibits two distinct transactions. The first derivative of this curveshows two peaks centered at 36 and 55° C., respectively, FIG. 2B. Thisdemonstrates that two distinct assemblies form and their meltingproperties, which derive from the oligonucleotide barcodes, can be usedto distinguish two protein targets.

In another aspect of the invention, a variation of the above aggregationmethod strategy can be used to increase the sensitivity of theaforementioned system and to increase the number of targets that can beinterrogated in one solution. See, for instance, FIG. 3 in Example 4.With this strategy, the protein targets can be detected indirectly viathe DNA biobarcodes or unique reporter oligonucleotides assigned tospecific target analytes. Generally, the suitable length, GC content,and sequence, and selection of the reporter oligonucleotide for thetarget analyte is predetermined prior to the assay. For instance, a12-mer oligonucleotide has 4¹² different sequences, many of which can beused to prepare a barcode for a polyvalent protein of interest as shownin FIG. 1A. In this variation of the assay, the melting properties ofthe aggregates that form are not measured in solution but rather thereporter oligonucleotides or DNA biobarcodes within the aggregates areseparated via centrifugation (e.g., 3000 rpm for 2 minutes) from theunreacted probes and target molecules. The aggregates are then denaturedby any suitable means, e.g., by adding water to the solution, to freethe reporter oligonucleotides or biobarcodes. If the reporteroligonucleotide is present in small amounts, it may be amplified bymethods known in the art. See, e.g., Sambrook et al., Molecular Cloning:A Laboratory Manual (2nd ed. 1989) and B. D. Hames and S. J. Higgins,Eds., Gene Probes 1 (IRL Press, New York, 1995). Preferred is polymerasechain reaction (PCR) amplification. The particles and proteins can beseparated from the reporter oligonucleotides by any suitable means,e.g., a centrifugal filter device (Millipore Microcon YM-100, 3500 rpmfor 25 min. Once the reporter oligonucleotides are isolated, they can becaptured on an oligonucleotide array and can be identified using one ofthe many suitable DNA detection assays (FIG. 3). For the examplesdescribed herein involving IgG1 and IgE, the reporter oligonucleotidesare captured on a microscope slide that has been functionalized witholigonucleotides (250 μm diameter spots) that are complementary to onehalf of the barcode of interest (A3 and B3 in FIG. 1). If the barcode iscaptured by the oligonucleotide array, a DNA-modified particle that iscomplementary to the remaining portion of the barcode can be hybridizedto the array (see experimental section). When developed via the standardscanometric approach ^([11]) (which involves treatment with photographicdeveloping solution), a flat bed scanner can be used to quantify theresults, FIG. 4.¹¹ If IgG1 is present, only the spot designed for IgG1shows measurable signal. Similarly if IgE is the only protein present,the spot designed for it only exhibits signal. Finally, if both proteinsare present, both spots exhibit intense signals.

The present invention is important because it provides two strategiesfor using nanoparticle probes (preferably gold nanoparticle probes),heavily functionalized with oligonucleotides, to detect single ormultiple polyvalent proteins in one solution. Indeed, the detection ofmultiple proteins in one sample is not trivial and often requires timeconsuming, expensive assay protocols. In this regard, others haverecently used fluorophore-labeled peptidonucleic acids and DNAmicroarrays to recognize multiple protein targets in one solution. ¹⁵⁻¹⁷However, this method relies on the binding of the proteins labeled witholigonucleotides to a microarray surface. The final step of the methoddescribed herein is based solely on the surface chemistry of ordinaryDNA. Therefore, it can incorporate many of the high sensitivity aspectsof state-of-the-art nanoparticle DNA detection methods, ^(9,11) butallows one to detect proteins rather than DNA without having theproteins present during the detection event. For surface assays,proteins are typically more difficult to work with than shortoligonucleotides because they tend to exhibit greater nonspecificbinding to solid supports, which often leads to higher backgroundsignals. Finally, for the homogeneous assay, the unusually sharp meltingprofiles associated with these nanoparticle structures will allow one todesign more biobarcodes than what would be possible with probes thatexhibit normal and broad DNA melting behavior.

The present invention contemplates the use of any suitable particlehaving oligonucleotides attached thereto that are suitable for use indetection assays. In practicing this invention, however, nanoparticlesare preferred. The size, shape and chemical composition of the particleswill contribute to the properties of the resulting probe including theDNA barcode. These properties include optical properties, optoelectronicproperties, electrochemical properties, electronic properties, stabilityin various solutions, pore and channel size variation, ability toseparate bioactive molecules while acting as a filter, etc. The use ofmixtures of particles having different sizes, shapes and/or chemicalcompositions, as well as the use of nanoparticles having uniform sizes,shapes and chemical composition, are contemplated. Examples of suitableparticles include, without limitation, nano- and microsized coreparticles, aggregate particles, isotropic (such as spherical particles)and anisotropic particles (such as non-spherical rods, tetrahedral,prisms) and core-shell particles such as the ones described in U.S.patent application Ser. No. 10/034,451, filed Dec. 28, 2002 andInternational application no. PCT/US01/50825, filed Dec. 28, 2002, whichare incorporated by reference in their entirety.

Nanoparticles useful in the practice of the invention include metal(e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe,CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g.,ferromagnetite) colloidal materials. Other nanoparticles useful in thepractice of the invention include ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS,PbSe, ZnTe, CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs. The sizeof the nanoparticles is preferably from about 5 nm to about 150 nm (meandiameter), more preferably from about 5 to about 50 nm, most preferablyfrom about 10 to about 30 nm. The nanoparticles may also be rods,prisms, or tetrahedra.

Methods of making metal, semiconductor and magnetic nanoparticles arewell-known in the art. See, e.g., Schmid, G. (ed.) Clusters and Colloids(VCH, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold: Principles,Methods, and Applications (Academic Press, San Diego, 1991); Massart,R., IEEE Taransactions On Magnetics, 17, 1247 (1981); Ahmadi, T. S. etal., Science, 272, 1924 (1996); Henglein, A. et al., J. Phys. Chem., 99,14129 (1995); Curtis, A. C., et al., Angew. Chem. Int. Ed. Engl., 27,1530 (1988).

Methods of making ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe,CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs nanoparticles arealso known in the art. See, e.g., Weller, Angew. Chem. Int. Ed. Engl.,32, 41 (1993); Henglein, Top. Curr. Chem., 143, 113 (1988); Henglein,Chem. Rev., 89, 1861 (1989); Brus, Appl. Phys. A., 53, 465 (1991);Bahncmann, in Photochemical Conversion and Storage of Solar Energy (eds.Pelizetti and Schiavello 1991), page 251; Wang and Herron, J. Phys.Chem., 95, 525 (1991); Olshavsky et al., J. Am. Chem. Soc., 112, 9438(1990); Ushida et al., J. Phys. Chem., 95, 5382 (1992).

Suitable nanoparticles are also commercially available from, e.g., TedPella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes, Inc.(gold).

Presently preferred for use in detecting nucleic acids are goldnanoparticles. Gold colloidal particles have high extinctioncoefficients for the bands that give rise to their beautiful colors.These intense colors change with particle size, concentration,interparticle distance, and extent of aggregation and shape (geometry)of the aggregates, making these materials particularly attractive forcolorimetric assays. For instance, hybridization of oligonucleotidesattached to gold nanoparticles with oligonucleotides and nucleic acidsresults in an immediate color change visible to the naked eye (see,e.g., the Examples).

The nanoparticles, the oligonucleotides or both are functionalized inorder to attach the oligonucleotides to the nanoparticles. Such methodsare known in the art. For instance, oligonucleotides functionalized withalkanethiols at their 3′-termini or 5′-termini readily attach to goldnanoparticles. See Whitesides, Proceedings of the Robert A. WelchFoundation 39th Conference On Chemical Research Nanophase Chemistry,Houston, Tex., pages 109-121 (1995). See also, Mucic et al. Chem.Commun. 555-557 (1996) (describes a method of attaching 3′ thiol DNA toflat gold surfaces; this method can be used to attach oligonucleotidesto nanoparticles). The alkanethiol method can also be used to attacholigonucleotides to other metal, semiconductor and magnetic colloids andto the other nanoparticles listed above. Other functional groups forattaching oligonucleotides to solid surfaces include phosphorothioategroups (see, e.g., U.S. Pat. No. 5,472,881 for the binding ofoligonucleotide-phosphorothioates to gold surfaces), substitutedalkylsiloxanes (see, e.g. Burwell, Chemical Technology, 4, 370-377(1974) and Matteucci and Caruthers, J. Am. Chem. Soc., 103, 3185-3191(1981) for binding of oligonucleotides to silica and glass surfaces, andGrabar et al., Anal. Chem., 67, 735-743 for binding ofaminoalkylsiloxanes and for similar binding of mercaptoaklylsiloxanes).Oligonucleotides terminated with a 5′ thionucleoside or a 3′thionucleoside may also be used for attaching oligonucleotides to solidsurfaces. The following references describe other methods which may beemployed to attached oligonucleotides to nanoparticles: Nuzzo et al., J.Am. Chem. Soc., 109, 2358 (1987) (disulfides on gold); Allara and Nuzzo,Langmuir, 1, 45 (1985) (carboxylic acids on aluminum); Allara andTompkins, J. Colloid Interface Sci., 49, 410-421 (1974) (carboxylicacids on copper); Iler, The Chemistry Of Silica, Chapter 6, (Wiley 1979)(carboxylic acids on silica); Timmons and Zisman, J. Phys. Chem., 69,984-990 (1965) (carboxylic acids on platinum); Soriaga and Hubbard, J.Am. Chem. Soc., 104, 3937 (1982) (aromatic ring compounds on platinum);Hubbard, Acc. Chem. Res., 13, 177 (1980) (sulfolanes, sulfoxides andother functionalized solvents on platinum); Hickman et al., J. Am. Chem.Soc., 111, 7271 (1989) (isonitriles on platinum); Maoz and Sagiv,Langmuir, 3, 1045 (1987) (silanes on silica); Maoz and Sagiv, Langmuir,3, 1034 (1987) (silanes on silica); Wasserman et al., Langmuir, 5, 1074(1989) (silanes on silica); Eltekova and Eltekov, Langmuir, 3, 951(1987) (aromatic carboxylic acids, aldehydes, alcohols and methoxygroups on titanium dioxide and silica); Lec et al., J. Phys. Chem., 92,2597 (1988) (rigid phosphates on metals).

U.S. patent application Ser. Nos. 09/760,500 and 09/820,279 andinternational application nos. PCT/US01/01190 and PCT/US01/10071describe oligonucleotides functionalized with a cyclic disulfide whichare useful in practicing this invention. The cyclic disulfidespreferably have 5 or 6 atoms in their rings, including the two sulfuratoms. Suitable cyclic disulfides are available commercially or may besynthesized by known procedures. The reduced form of the cyclicdisulfides can also be used.

Preferably, the linker further comprises a hydrocarbon moiety attachedto the cyclic disulfide. Suitable hydrocarbons are availablecommercially, and are attached to the cyclic disulfides Preferably thehydrocarbon moiety is a steroid residue. Oligonucleotide-nanoparticleconjugates prepared using linkers comprising a steroid residue attachedto a cyclic disulfide have unexpectedly been found to be remarkablystable to thiols (e.g., dithiothreitol used in polymerase chain reaction(PCR) solutions) as compared to conjugates prepared using alkanethiolsor acyclic disulfides as the linker. Indeed, theoligonucleotide-nanoparticle conjugates of the invention have been foundto be 300 times more stable. This unexpected stability is likely due tothe fact that each oligonucleotide is anchored to a nanoparticle throughtwo sulfur atoms, rather than a single sulfur atom. In particular, it isthought that two adjacent sulfur atoms of a cyclic disulfide would havea chelation effect which would be advantageous in stabilizing theoligonucleotide-nanoparticle conjugates. The large hydrophobic steroidresidues of the linkers also appear to contribute to the stability ofthe conjugates by screening the nanoparticles from the approach ofwater-soluble molecules to the surfaces of the nanoparticles.

In view of the foregoing, the two sulfur atoms of the cyclic disulfideshould preferably be close enough together so that both of the sulfuratoms can attach simultaneously to the nanoparticle. Most preferably,the two sulfur atoms are adjacent each other. Also, the hydrocarbonmoiety should be large so as to present a large hydrophobic surfacescreening the surfaces of the nanoparticles.

The oligonucleotide-cyclic nanoparticle conjugates that employ cyclicdisulfide linkers may be used as probes in diagnostic assays fordetecting target analytes in a sample as described in U.S. patentapplication Ser. Nos. 09/760,500 and 09/820,279 and internationalapplication nos. PCT/US01/01190 and PCT/US01/10071. These conjugateshave been found to improve the sensitivity of diagnostic assays in whichthey are used. In particular, assays employingoligonucleotide-nanoparticle conjugates prepared using linkerscomprising a steroid residue attached to a cyclic disulfide have beenfound to be about 10 times more sensitive than assays employingconjugates prepared using alkanethiols or acyclic disulfides as thelinker.

Each nanoparticle will have a plurality of oligonucleotides attached toit. As a result, each nanoparticle-oligonucleotide conjugate can bind toa plurality of oligonucleotides or nucleic acids having thecomplementary sequence.

Oligonucleotides of defined sequences are used for a variety of purposesin the practice of the invention. Methods of making oligonucleotides ofa predetermined sequence are well-known. See, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual (2nd ed. 1989) and F. Eckstein(ed.) Oligonucleotides and Analogues, 1st Ed. (Oxford University Press,New York, 1991). Solid-phase synthesis methods are preferred for botholigoribonucleotides and oligodeoxyribonucleotides (the well-knownmethods of synthesizing DNA are also useful for synthesizing RNA).Oligoribonucleotides and oligodeoxyribonucleotides can also be preparedenzymatically. For oligonucleotides having bound thereto a specificbinding complement to a target analyte, any suitable method forattaching the specific binding complement such as proteins to theoligonucleotide may be used.

Any suitable method for attaching oligonucleotides onto the nanospheresurface may be used. A particularly preferred method for attachingoligonucleotides onto a surface is based on an aging process describedin U.S. application Ser. No. 09/344,667, filed Jun. 25, 1999; Ser. No.09/603,830, filed Jun. 26, 2000; Ser. No. 09/760,500, filed Jan. 12,2001; Ser. No. 09/820,279, filed Mar. 28, 2001; Ser. No. 09/927,777,filed Aug. 10, 2001; and in International application nos.PCT/US97/12783, filed Jul. 21, 1997; PCT/US00/17507, filed Jun. 26,2000; PCT/US01/01190, filed Jan. 12, 2001; PCT/US01/10071, filed Mar.28, 2001, the disclosures which are incorporated by reference in theirentirety. The aging process provides nanoparticle-oligonucleotideconjugates with unexpected enhanced stability and selectivity. Themethod comprises providing oligonucleotides preferably having covalentlybound thereto a moiety comprising a functional group which can bind tothe nanoparticles. The moieties and functional groups are those thatallow for binding (i.e., by chemisorption or covalent bonding) of theoligonucleotides to nanoparticles. For instance, oligonucleotides havingan alkanethiol, an alkanedisulfide or a cyclic disulfide covalentlybound to their 5′ or 3′ ends can be used to bind the oligonucleotides toa variety of nanoparticles, including gold nanoparticles.

The oligonucleotides are contacted with the nanoparticles in water for atime sufficient to allow at least some of the oligonucleotides to bindto the nanoparticles by means of the functional groups. Such times canbe determined empirically. For instance, it has been found that a timeof about 12-24 hours gives good results. Other suitable conditions forbinding of the oligonucleotides can also be determined empirically. Forinstance, a concentration of about 10-20 nM nanoparticles and incubationat room temperature gives good results.

Next, at least one salt is added to the water to form a salt solution.The salt can be any suitable water-soluble salt. For instance, the saltmay be sodium chloride, magnesium chloride, potassium chloride, ammoniumchloride, sodium acetate, ammonium acetate, a combination of two or moreof these salts, or one of these salts in phosphate buffer. Preferably,the salt is added as a concentrated solution, but it could be added as asolid. The salt can be added to the water all at one time or the salt isadded gradually over time. By “gradually over time” is meant that thesalt is added in at least two portions at intervals spaced apart by aperiod of time. Suitable time intervals can be determined empirically.

The ionic strength of the salt solution must be sufficient to overcomeat least partially the electrostatic repulsion of the oligonucleotidesfrom each other and, either the electrostatic attraction of thenegatively-charged oligonucleotides for positively-chargednanoparticles, or the electrostatic repulsion of the negatively-chargedoligonucleotides from negatively-charged nanoparticles. Graduallyreducing the electrostatic attraction and repulsion by adding the saltgradually over time has been found to give the highest surface densityof oligonucleotides on the nanoparticles. Suitable ionic strengths canbe determined empirically for each salt or combination of salts. A finalconcentration of sodium chloride of from about 0.1 M to about 1.0 M inphosphate buffer, preferably with the concentration of sodium chloridebeing increased gradually over time, has been found to give goodresults.

After adding the salt, the oligonucleotides and nanoparticles areincubated in the salt solution for an additional period of timesufficient to allow sufficient additional oligonucleotides to bind tothe nanoparticles to produce the stable nanoparticle-oligonucleotideconjugates. As will be described in detail below, an increased surfacedensity of the oligonucleotides on the nanoparticles has been found tostabilize the conjugates. The time of this incubation can be determinedempirically. A total incubation time of about 24-48, preferably 40hours, has been found to give good results (this is the total time ofincubation; as noted above, the salt concentration can be increasedgradually over this total time). This second period of incubation in thesalt solution is referred to herein as the “aging” step. Other suitableconditions for this “aging” step can also be determined empirically. Forinstance, incubation at room temperature and pH 7.0 gives good results.

The conjugates produced by use of the “aging” step have been found to beconsiderably more stable than those produced without the “aging” step.As noted above, this increased stability is due to the increased densityof the oligonucleotides on the surfaces of the nanoparticles which isachieved by the “aging” step. The surface density achieved by the“aging” step will depend on the size and type of nanoparticles and onthe length, sequence and concentration of the oligonucleotides. Asurface density adequate to make the nanoparticles stable and theconditions necessary to obtain it for a desired combination ofnanoparticles and oligonucleotides can be determined empirically.Generally, a surface density of at least 10 picomoles/cm² will beadequate to provide stable nanoparticle-oligonucleotide conjugates.Preferably, the surface density is at least 15 picomoles/cm². Since theability of the oligonucleotides of the conjugates to hybridize withnucleic acid and oligonucleotide targets can be diminished if thesurface density is too great, the surface density is preferably nogreater than about 35-40 picomoles/cm².

As used herein, “stable” means that, for a period of at least six monthsafter the conjugates are made, a majority of the oligonucleotides remainattached to the nanoparticles and the oligonucleotides are able tohybridize with nucleic acid and oligonucleotide targets under standardconditions encountered in methods of detecting nucleic acid and methodsof nanofabrication.

It has been found that the hybridization efficiency ofnanoparticle-oligonucleotide conjugates can be increased dramatically bythe use of recognition oligonucleotides which comprise a recognitionportion and a spacer portion. “Recognition oligonucleotides” areoligonucleotides which comprise a sequence complementary to at least aportion of the sequence of a nucleic acid or oligonucleotide target. Inthis embodiment, the recognition oligonucleotides comprise a recognitionportion and a spacer portion, and it is the recognition portion whichhybridizes to the nucleic acid or oligonucleotide target. The spacerportion of the recognition oligonucleotide is designed so that it canbind to the nanoparticles. For instance, the spacer portion could have amoiety covalently bound to it, the moiety comprising a functional groupwhich can bind to the nanoparticles. These are the same moieties andfunctional groups as described above. As a result of the binding of thespacer portion of the recognition oligonucleotide to the nanoparticles,the recognition portion is spaced away from the surface of thenanoparticles and is more accessible for hybridization with its target.The length and sequence of the spacer portion providing good spacing ofthe recognition portion away from the nanoparticles can be determinedempirically. It has been found that a spacer portion comprising at leastabout 10 nucleotides, preferably 10-30 nucleotides, gives good results.The spacer portion may have any sequence which does not interfere withthe ability of the recognition oligonucleotides to become bound to thenanoparticles or to a nucleic acid or oligonucleotide target. Forinstance, the spacer portions should not sequences complementary to eachother, to that of the recognition olignucleotides, or to that of thenucleic acid or oligonucleotide target of the recognitionoligonucleotides. Preferably, the bases of the nucleotides of the spacerportion are all adenines, all thymines, all cytidines, or all guanines,unless this would cause one of the problems just mentioned. Morepreferably, the bases are all adenines or all thymines. Most preferablythe bases are all thymines.

It has further been found that the use of diluent oligonucleotides inaddition to recognition oligonucleotides provides a means of tailoringthe conjugates to give a desired level of hybridization. The diluent andrecognition oligonucleotides have been found to attach to thenanoparticles in about the same proportion as their ratio in thesolution contacted with the nanoparticles to prepare the conjugates.Thus, the ratio of the diluent to recognition oligonucleotides bound tothe nanoparticles can be controlled so that the conjugates willparticipate in a desired number of hybridization events. The diluentoligonucleotides may have any sequence which does not interfere with theability of the recognition oligonucleotides to be bound to thenanoparticles or to bind to a nucleic acid or oligonucleotide target.For instance, the diluent oligonulceotides should not have a sequencecomplementary to that of the recognition olignucleotides or to that ofthe nucleic acid or oligonucleotide target of the recognitionoligonucleotides. The diluent oligonucleotides are also preferably of alength shorter than that of the recognition oligonucleotides so that therecognition oligonucleotides can bind to their nucleic acid oroligonucleotide targets. If the recognition oligonucleotides comprisespacer portions, the diluent oligonulceotides are, most preferably,about the same length as the spacer portions. In this manner, thediluent oligonucleotides do not interefere with the ability of therecognition portions of the recognition oligonucleotides to hybridizewith nucleic acid or oligonucleotide targets. Even more preferably, thediluent oligonucleotides have the same sequence as the sequence of thespacer portions of the recognition oligonucleotides.

For detection of the presence of a target analyte in a sample, particlecomplex probes, preferably nanoparticle complex probes, are used. Theseparticle complexes may be generated prior to conducting the actual assayor in situ while conducting the assay. These complexes comprise aparticle, preferably a nanoparticle, having oligonucleotides boundthereto, a reporter oligonucleotide, and an oligonucleotide having boundthereto a specific binding complement of a target analyte. The DNAbarcode or reporter oligonucleotides has a sequence having at least twoportions and joins via hybridization the nanoparticle havingoligonucleotides bound thereto and the oligonucleotide having boundthereto the specific binding complement. The oligonucleotides bound tothe nanoparticles have a sequence that is complementary to one portionof the reporter oligonucleotide and the oligonucleotide having boundthereto the specific binding complement having a sequence that iscomplementary to a second portion of the reporter oligonucleotide. Thereporter oligonucleotides have at least two portions and joins viahybridization the nanoparticle having oligonucleotides bound thereto andthe oligonucleotide having bound thereto the specific bindingcomplement. When employed in a sample containing the target analyte, thenanoparticle complex binds to the target analyte and aggregation occurs.The aggregates may be isolated and subject to further melting analysisto identify the particular target analyte where multiple targets arepresent as discussed above. Alternatively, the aggregates can bedehybridized to release the reporter oligonucleotides. These reporteroligonucleotides can then be detected by any suitable DNA detectionsystem using any suitable detection probes.

In another aspect of the invention, the reporter oligonucleotidesreleased by dehybridization of the aggregates can be detected using asubstrate having oligonucleotides bound thereto. The oligonucleotideshave a sequence complementary to at least one portion of the reporteroligonucleotides. Some embodiments of the method of detecting thereporter oligonucleotides utilize a substrate having complementaryoligonucleotides bound thereto to capture the reporter oligonucleotides.These captured reporter oligonucleotides are then detected by anysuitable means. By employing a substrate, the detectable change (thesignal) can be amplified and the sensitivity of the assay increased.

Any substrate can be used which allows observation of the detectablechange. Suitable substrates include transparent solid surfaces (e.g.,glass, quartz, plastics and other polymers), opaque solid surface (e.g.,white solid surfaces, such as TLC silica plates, filter paper, glassfiber filters, cellulose nitrate membranes, nylon membranes), andconducting solid surfaces (e.g., indium-tin-oxide (ITO)). The substratecan be any shape or thickness, but generally will be flat and thin.Preferred are transparent substrates such as glass (e.g., glass slides)or plastics (e.g., wells of microtiter plates).

Any suitable method for attaching oligonucleotides to a substrate may beused. For instance, oligonucleotides can be attached to the substratesas described in, e.g., Chrisey et al., Nucleic Acids Res., 24, 3031-3039(1996); Chrisey et al., Nucleic Acids Res., 24, 3040-3047 (1996); Mucicet al., Chem. Commun., 555 (1996); Zimmermann and Cox, Nucleic AcidsRes., 22, 492 (1994); Bottomley et al., J. Vac. Sci. Technol. A, 10, 591(1992); and Hegner et al., FEBS Lett., 336, 452 (1993).

The oligonucleotides attached to the substrate have a sequencecomplementary to a first portion of the sequence of reporteroligonucleotides to be detected. The reporter oligonucleotide iscontacted with the substrate under conditions effective to allowhybridization of the oligonucleotides on the substrate with the reporteroligonucleotide. In this manner the reporter oligonucleotide becomesbound to the substrate. Any unbound reporter oligonucleotide ispreferably washed from the substrate before adding a detection probesuch as nanoparticle-oligonucleotide conjugates.

In one aspect of the invention, the reporter oligonucleotide bound tothe oligonucleotides on the substrate is contacted with a first type ofnanoparticles having oligonucleotides attached thereto. Theoligonucleotides have a sequence complementary to a second portion ofthe sequence of the reporter oligonucleotide, and the contacting takesplace under conditions effective to allow hybridization of theoligonucleotides on the nanoparticles with the reporter oligonucleotide.In this manner the first type of nanoparticles become bound to thesubstrate. After the nanoparticle-oligonucleotide conjugates are boundto the substrate, the substrate is washed to remove any unboundnanoparticle-oligonucleotide conjugates.

The oligonucleotides on the first type of nanoparticles may all have thesame sequence or may have different sequences that hybridize withdifferent portions of the reporter oligonucleotide to be detected. Whenoligonucleotides having different sequences are used, each nanoparticlemay have all of the different oligonucleotides attached to it or,preferably, the different oligonucleotides are attached to differentnanoparticles. Alternatively, the oligonucleotides on each of the firsttype of nanoparticles may have a plurality of different sequences, atleast one of which must hybridize with a portion of the reporteroligonucleotide to be detected.

Optionally, the first type of nanoparticle-oligonucleotide conjugatesbound to the substrate is contacted with a second type of nanoparticleshaving oligonucleotides attached thereto. These oligonucleotides have asequence complementary to at least a portion of the sequence(s) of theoligonucleotides attached to the first type of nanoparticles, and thecontacting takes place under conditions effective to allow hybridizationof the oligonucleotides on the first type of nanoparticles with those onthe second type of nanoparticles. After the nanoparticles are bound, thesubstrate is preferably washed to remove any unboundnanoparticle-oligonucleotide conjugates.

The combination of hybridizations produces a detectable change. Thedetectable changes are the same as those described above, except thatthe multiple hybridizations result in an amplification of the detectablechange. In particular, since each of the first type of nanoparticles hasmultiple oligonucleotides (having the same or different sequences)attached to it, each of the first type of nanoparticle-oligonucleotideconjugates can hybridize to a plurality of the second type ofnanoparticle-oligonucleotide conjugates. Also, the first type ofnanoparticle-oligonucleotide conjugates may be hybridized to more thanone portion of the reporter oligonucleotide to be detected. Theamplification provided by the multiple hybridizations may make thechange detectable for the first time or may increase the magnitude ofthe detectable change. This amplification increases the sensitivity ofthe assay, allowing for detection of small amounts of reporteroligonucleotide.

If desired, additional layers of nanoparticles can be built up bysuccessive additions of the first and second types ofnanoparticle-oligonucleotide conjugates. In this way, the number ofnanoparticles immobilized per molecule of target nucleic acid can befurther increased with a corresponding increase in intensity of thesignal.

Also, instead of using first and second types ofnanoparticle-oligonucleotide conjugates designed to hybridize to eachother directly, nanoparticles bearing oligonucleotides that would serveto bind the nanoparticles together as a consequence of hybridizationwith binding oligonucleotides could be used.

When a substrate is employed, a plurality of the initial types ofnanoparticle-oligonucleotide conjugates or oligonucleotides can beattached to the substrate in an array for detecting multiple portions ofa target reporter oligonucleotide, for detecting multiple differentreporter oligonucleotides, or both. For instance, a substrate may beprovided with rows of spots, each spot containing a different type ofoligonucleotide designed to bind to a portion of a target reporteroligonucleotide. A sample containing one or more reporteroligonucleotides is applied to each spot, and the rest of the assay isperformed in one of the ways described above using appropriateoligonucleotide-nanoparticle conjugates.

Finally, when a substrate is employed, a detectable change can beproduced or further enhanced by silver staining. Silver staining can beemployed with any type of nanoparticles that catalyze the reduction ofsilver. Preferred are nanoparticles made of noble metals (e.g., gold andsilver). See Bassell, et al., J. Cell Biol., 126, 863-876 (1994);Braun-Howland et al., Biotechniques, 13, 928-931 (1992). If thenanoparticles being employed for the detection of a nucleic acid do notcatalyze the reduction of silver, then silver ions can be complexed tothe nucleic acid to catalyze the reduction. See Braun et al., Nature,391, 775 (1998). Also, silver stains are known which can react with thephosphate groups on nucleic acids.

Silver staining can be used to produce or enhance a detectable change inany assay performed on a substrate, including those described above. Inparticular, silver staining has been found to provide a huge increase insensitivity for assays employing a single type of nanoparticle so thatthe use of layers of nanoparticles can often be eliminated.

In assays for detecting reporter oligonucleotides performed on asubstrate, the detectable change can be observed with an opticalscanner. Suitable scanners include those used to scan documents into acomputer which are capable of operating in the reflective mode (e.g., aflatbed scanner), other devices capable of performing this function orwhich utilize the same type of optics, any type of greyscale-sensitivemeasurement device, and standard scanners which have been modified toscan substrates according to the invention (e.g., a flatbed scannermodified to include a holder for the substrate) (to date, it has notbeen found possible to use scanners operating in the transmissive mode).The resolution of the scanner must be sufficient so that the reactionarea on the substrate is larger than a single pixel of the scanner. Thescanner can be used with any substrate, provided that the detectablechange produced by the assay can be observed against the substrate(e.g., a grey spot, such as that produced by silver staining, can beobserved against a white background, but cannot be observed against agrey background). The scanner can be a black-and-white scanner or,preferably, a color scanner. Most preferably, the scanner is a standardcolor scanner of the type used to scan documents into computers. Suchscanners are inexpensive and readily available commercially. Forinstance, an Epson Expression 636 (600×600 dpi), a UMAX Astra 1200(300×300 dpi), or a Microtec 1600 (1600×1600 dpi) can be used. Thescanner is linked to a computer loaded with software for processing theimages obtained by scanning the substrate. The software can be standardsoftware which is readily available commercially, such as AdobePhotoshop 5.2 and Corel Photopaint 8.0. Using the software to calculategreyscale measurements provides a means of quantitating the results ofthe assays. The software can also provide a color number for coloredspots and can generate images (e.g., printouts) of the scans which canbe reviewed to provide a qualitative determination of the presence of anucleic acid, the quantity of a nucleic acid, or both. The computer canbe a standard personal computer which is readily available commercially.Thus, the use of a standard scanner linked to a standard computer loadedwith standard software can provide a convenient, easy, inexpensive meansof detecting and quantitating nucleic acids when the assays areperformed on substrates. The scans can also be stored in the computer tomaintain a record of the results for further reference or use. Ofcourse, more sophisticated instruments and software can be used, ifdesired.

A universal nanoparticle-oligonucleotide conjugate which may be used inan assay for any target reporter oligonucleotide. This “universal probe”has oligonucleotides of a single sequence attached to it and iscomplementary with a portion of the reporter oligonucleotide. Theseoligonucleotides bound to the universal probe can hybridize with aportion of the reporter oligonucleotides bound to the support. The firstportion is complementary to at least a portion of the sequence of theoligonucleotides on the nanoparticles. The second portion iscomplementary to a portion of the sequence of the nucleic acid to bedetected. A plurality of binding oligonucleotides having the same firstportion and different second portions can be used, in which case the“universal probe”, after hybridization to the binding oligonucleotides,can bind to multiple portions of the nucleic acid to be detected or todifferent nucleic acid targets.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity. For example, “a characteristic” refers to one or morecharacteristics or at least one characteristic. As such, the terms “a”(or “an”), “one or more” and “at least one” are used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” have been used interchangeably.

EXAMPLES Example 1 Preparation of Oligonucleotide-Modified GoldNanoparticles A. Preparation Of Gold Nanoparticles

Oligonucleotide-modified 13 nm Au particles were prepared by literaturemethods (˜110 oligonucleotides/particle)¹⁸⁻²⁰. Gold colloids (13 nmdiameter) were prepared by reduction of HAuCl₄ with citrate as describedin Frens, Nature Phys. Sci., 241, 20 (1973) and Grabar, Anal. Chem., 67,735 (1995). Briefly, all glassware was cleaned in aqua regia (3 partsHCl, 1 part HNO₃), rinsed with Nanopure H₂O, then oven dried prior touse. HAuCl₄ and sodium citrate were purchased from Aldrich ChemicalCompany. An aqueous solution of HAuCl₄ (1 mM, 500 mL) was brought to areflux while stirring, and then 50 mL of a 38.8 mM trisodium citratesolution was added quickly, which resulted in a change in solution colorfrom pale yellow to deep red. After the color change, the solution wasrefluxed for an additional fifteen minutes, allowed to cool to roomtemperature, and subsequently filtered through a Micron Separations Inc.0.45 micron nylon filter. Au colloids were characterized by UV-visspectroscopy using a Hewlett Packard 8452A diode array spectrophotometerand by Transmission Electron Microscopy (TEM) using a Hitachi 8100transmission electron microscope. A typical solution of 13 nm diametergold particles exhibited a characteristic surface plasmon band centeredat 518-520 nm. Gold particles with diameters of 13 nm will produce avisible color change when aggregated with target and probeoligonucleotide sequences in the 10-72 nucleotide range.

B. Synthesis Of Oligonucleotides

Oligonucleotides were synthesized on a 1 micromole scale using aMilligene Expedite DNA synthesizer in single column mode usingphosphoramidite chemistry. Eckstein, F. (ed.) Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991). All solutionswere purchased from Milligene (DNA synthesis grade). Average couplingefficiency varied from 98 to 99.8%, and the final dimethoxytrityl (DMT)protecting group was not cleaved from the oligonucleotides to aid inpurification.

For 3′-thiol-oligonucleotides, Thiol-Modifier C3 S-S CPG support waspurchased from Glen Research and used in the automated synthesizer. Thefinal dimethoxytrityl (DMT) protecting group was not removed to aid inpurification. After synthesis, the supported oligonucleotide was placedin 1 mL of concentrated ammonium hydroxide for 16 hours at 55° C. tocleave the oligonucleotide from the solid support and remove theprotecting groups from the bases.

After evaporation of the ammonia, the oligonucleotides were purified bypreparative reverse-phase HPLC using an HP ODS Hypersil column (5 μm,250×4 mm) with 0.03 M triethyl ammonium acetate (TEAA), pH 7 and a1%/minute gradient of 95% CH₃CN/5% 0.03 M TEAA at a flow rate of 1mL/minute, while monitoring the UV signal of DNA at 254 nm. Theretention time of the DMT protected modified 12-base oligomer was 30minutes. The DMT was subsequently cleaved by soaking the purifiedoligonucleotide in an 80% acetic acid solution for 30 minutes, followedby evaporation; the oligonucleotide was redispersed in 500 μL of water,and the solution was extracted with ethyl acetate (3×300 μL). Afterevaporation of the solvent, the oligonucleotide (10 OD's) wasredispersed in 100 υL of a 0.04 M DTT, 0.17 M phosphate buffer (pH 8)solution overnight at 50° C. to cleave the 3′ disulfide. Aliquots ofthis solution (<10 OD's) were purified through a desalting NAP-5 column.The amount of oligonucleotide was determined by absorbance at 260 nm.Purity was assessed by ion-exchange HPLC using a Dionex Nucleopac PA-100column (250×4 mm) with 10 mM NaOH (pH 12) and a 2%/minute gradient of 10mM NaOH, 1 M NaCl at a flow rate of 1 mL/minute while monitoring the UVsignal of DNA at 254 nm. Three peaks with retention times (T_(r)) of18.5, 18.9 and 22 minutes were observed. The main single peak atT_(r)=22.0 minutes, which has been attributed to the disulfide, was 79%by area. The two peaks with shorter retention times of 18.5 and 18.9minutes were ˜9% and 12% by area respectively, and have been attributedto oxidized impurity and residual thiol oligonucleotide.

5′-Alkylthiol modified oligonucleotides were prepared using thefollowing protocol: 1) a CPG-bound, detritylated oligonucleotide wassynthesized on an automated DNA synthesizer (Expedite) using standardprocedures; 2) the CPG-cartridge was removed and disposable syringeswere attached to the ends; 3) 200 μL of a solution containing 20 μmoleof 5-Thiol-Modifier C6-phosphoramidite (Glen Research) in dryacetonitrile was mixed with 200 μL of standard “tetrazole activatorsolution” and, via one of the syringes, introduced into the cartridgecontaining the oligonucleotide-CPG; 4) the solution was slowly pumpedback and forth through the cartridge for 10 minutes and then ejectedfollowed by washing with dry acetonitrile (2×1 mL); 5) the intermediatephosphite was oxidized with 700 μL of 0.02 M iodine inTHF/pyridine/water (30 seconds) followed by washing withacetonitrile/pyridine (1:1; 2×1 mL) and dry acetonitirile. Thetrityloligonucleotide derivative was then isolated and purified asdescribed by the 3′-alkylthiol oligonucleotides; then the tritylprotecting group was cleaved by adding 15 υL (for 10 OD's) of a 50 mMAgNO₃ solution to the dry oligonucleotide sample for 20 minutes, whichresulted in a milky white suspension. The excess silver nitrate wasremoved by adding 20 ΦL of a 10 mg/mL solution of DTT (five minutereaction time), which immediately formed a yellow precipitate that wasremoved by centrifugation. Aliquots of the oligonucleotide solution (<10OD's) were then transferred onto a desalting NAP-5 column forpurification. The final amount and the purity of the resulting 5′alkylthiol oligonucleotides were assessed using the techniques describedabove for 3′ alkylthiol oligonucleotides. Two major peaks were observedby ion-exchange HPLC with retention times of 19.8 minutes (thiol peak,16% by area) and 23.5 minutes (disulfide peak, 82% by area).

C. Attachment of Oligonucleotides to Gold Nanoparticles

A 1 mL solution of the gold colloids (17 nM) in water was mixed withexcess (3.68 υM) thiol-oligonucleotide (22 bases in length) in water,and the mixture was allowed to stand for 12-24 hours at roomtemperature. Then, the solution was brought to 0.1 M NaCl, 10 mMphosphate buffer (pH 7) and allowed to stand for 40 hours. This “aging”step was designed to increase the surface coverage by thethiol-oligonucleotides and to displace oligonucleotide bases from thegold surface. The solution was next centrifuged at 14,000 rpm in anEppendorf Centrifuge 5414 for about 25 minutes to give a very pale pinksupernatant containing most of the oligonucleotide (as indicated by theabsorbance at 260 nm) along with 7-10% of the colloidal gold (asindicated by the absorbance at 520 nm), and a compact, dark, gelatinousresidue at the bottom of the tube. The supernatant was removed, and theresidue was resuspended in about 200 μL of buffer (10 mM phosphate, 0.1M NaCl) and recentrifuged. After removal of the supernatant solution,the residue was taken up in 1.0 mL of buffer (10 mM phosphate, 0.3 MNaCl, 0.01% NaN₃). The resulting red master solution was stable (i.e.,remained red and did not aggregate) on standing for months at roomtemperature, on spotting on silica thin-layer chromatography (TLC)plates (see Example 4), and on addition to 1 M NaCl, 10 mM MgCl₂, orsolutions containing high concentrations of salmon sperm DNA.

Example 2 Preparation of Hapten-Modified Oligonucleotides

Hapten-modified oligonucleotides were prepared with a biotin-triethyleneglycol phosphoramidite for A1 and 2,4-dinitrophenyl-triethylene glycolphosphoramidite for B1 (Glen research) using standard solid-phase DNAsynthesis procedures.²¹

Biotin modified oligonucleotides were prepared using the followingprotocol: A CPG-bound, detritylated oligonucleotide was synthesized onan automated DNA synthesizer (Expedite) using standard procedures²¹. TheCPG-cartridge was then removed and disposable syringes were attached tothe ends. 200 μL of a solution containing 20 μmole of biotin-triethyleneglycol phosphoramidite in dry acetonitrile was then mixed with 200 μL ofstandard “tetrazole activator solution” and, via one of the syringes,introduced into the cartridge containing the oligonucleotide-CPG. Thesolution then was slowly pumped back and forth through the cartridge for10 minutes and then ejected followed by washing with dry acetonitrile(2×1 mL). Thereafter, the intermediate phosphite was oxidized with 700μL of 0.02 M iodine in THF/pyridine/water (30 seconds) followed bywashing with acetonitrile/pyridine (1:1; 2×1 mL) and dry acetonitirilewith subsequent drying of the column with a stream of nitrogen. Thetrityl protecting group was not removed, which aids in purification. Thesupported oligonucleotide was placed in 1 mL of concentrated ammoniumhydroxide for 16 hours at 55° C. to cleave the oligonucleotide from thesolid support and remove the protecting groups from the bases. Afterevaporation of the ammonia, the oligonucleotides were purified bypreparative reverse-phase HPLC using an HP ODS Hypersil column (5 μm,250×4 mm) with 0.03 M triethyl ammonium acetate (TEAA), pH 7 and a1%/minute gradient of 95% CH₃CN/5% 0.03 M TEAA at a flow rate of 1mL/minute, while monitoring the UV signal of DNA at 254 nm. Theretention time of the DMT protected oligonucleotides was 32 minutes. TheDMT was subsequently cleaved by soaking the purified oligonucleotide inan 80% acetic acid solution for 30 minutes, followed by evaporation; theoligonucleotide was redispersed in 500 μL of water, and the solution wasextracted with ethyl acetate (3×300 μL) and dried. The same protocol wasused to synthesize DNP modified oligonucleotide using2,4-dinitrophenyl-triethylene glycol phosphoramidite.

Example 3 Assay Using Nanoparticle Complex Probes

The Oligonucleotide-modified 13 nm gold particles were prepared asdescribed in Example 1. Hapten-modified oligonucleotides were preparedas described in Example 2 with a biotin-triethylene glycolphosphoramidite for A1 and 2,4-dinitrophenyl-triethylene glycolphosphoramidite for B1 (Glen research) using standard solid-phase DNAsynthesis procedures.²¹ The PBS buffer solution used in this researchconsists of 0.3 M NaCl and 10 mM phosphate buffer (pH 7). IgE and IgG1were purchased from Sigma Aldrich (Milwaukee, Wis.) and dissolved in 0.3M PBS buffer with 0.05% Tween 20 (final concentration: 4.3×10⁻⁸ b/μl)and background proteins (10 ug/ml of lysozyme, 1% bovine serum albumin,and 5.3 ug/mil of anti-digoxin; 10 uL of each) prior to use.

To prepare the probes, the oligonucleotide modified particles (13 nM,300 μL) were hybridized with hapten-modified complementaryoligonucleotides (10 μL of 10 μM) and biobarcode DNA (10 μL of 10 μM) atroom temperature for 2-3 h, sequences given in FIG. 1. Unreactedhapten-modified oligonucleotide and biobarcodes were removed viacentrifugation (12,000 rpm, 20 min) of the nanoparticle probes andsubsequent decanting of the supernatant.

In a typical assay for IgE and/or IgG1, the target proteins (40 μl of 43μg/ml for each) were added to the solution containing the probes (˜13nM), and the mixture was incubated at 37° C. for 50 minutes tofacilitate protein-hapten complexation. To ensure complete reactionamong all the components, especially the complementary DNA strands, thesolution was incubated to expedite hybridization at −15° C. for 20minutes (Boekel Tropicooler Hot/Cold Block Incubator) and stored at 4°C. for 24 hours. If the target protein is present, particle aggregationtakes place effecting a shift in the gold nanoparticle plasmon band anda red-to-purple color change along with precipitation. The hybridizedproducts were centrifuged (3000 rpm for 2 minutes), and the supernatantcontaining unreacted elements was decanted prior to analysis. Todetermine which proteins are present, a melting analysis which monitorsthe extinction at 260 nm as a function of temperature is carried out inthe solution, FIG. 2. When IgG1 is treated with the probes via theaforementioned protocol, the solution turns pinkish-blue, indicating theformation of nanoparticle aggregates. In a control experiment where notarget but background proteins are present, there is no discernibleprecipitation. A melting analysis of the solution shows a sharptransition with a melting temperature (Tm) of 55° C. This is theexpected transition for the IgG1 target, FIG. 2A (---). If IgE is addedto a fresh solution of probes, the same color change is observed but themelting analysis provides a curve with a Tm of 36° C., the expectedtransition for this target, FIG. 2A (

). Significantly, when both protein targets are added to the solution ofprobes, the solution turns dark purple, and the melting analysisexhibits two distinct transactions. The first derivative of this curveshows two peaks centered at 36 and 55° C., respectively, FIG. 2B. Thisdemonstrates that two distinct assemblies form and their meltingproperties, which derive from the oligonucleotide barcodes, can be usedto distinguish two protein targets.

Example 4 Assay Using Nanoparticle Complex Probes

A variation of this strategy can be used to increase the sensitivity ofthe aforementioned system and to increase the number of targets that canbe interrogated in one solution (FIG. 3). With this strategy, theprotein targets can be detected indirectly via the DNA biobarcodes. A12-mer oligonucleotide has 4¹² different sequences, many of which can beused to prepare a barcode for a polyvalent protein of interest via FIG.1A. In this variation of the assay, the melting properties of theaggregates that form are not measured in solution but rather the DNAbiobarcodes within the aggregates are separated via centrifugation (3000rpm fro 2 minutes) from the unreacted probes and target molecules. Theaggregates are then denatured by adding water to the solution, freeingthe complexed DNA. The particles and proteins can be separated from theDNA barcodes with a centrifugal filter device (Millipore MicroconYM-100, 3500 rpm for 25 min). Once the DNA barcodes are isolated, theycan be captured on an oligonucleotide array and can be identified usingone of the many DNA detection assays (FIG. 3). For the examplesdescribed herein involving IgG1 and IgE, the barcodes are captured on amicroscope slide that has been functionalized with oligonucleotides (250μm diameter spots) that are complementary to one half of the barcode ofinterest (A3 and B3 in FIG. 1). If the barcode is captured by theoligonucleotide array, a DNA-modified particle that is complementary tothe remaining portion of the barcode can be hybridized to the array (seeexperimental section). When developed via the standard scanometricapproach ^([11]) (which involves treatment with photographic developingsolution), a flat bed scanner can be used to quantify the results, FIG.4.¹¹ If IgG1 is present, only the spot designed for IgG1 showsmeasurable signal. Similarly if IgE is the only protein present, thespot designed for it only exhibits signal. Finally, if both proteins arepresent, both spots exhibit intense signals.

For scanometric DNA biobarcode detection, the DNA/Au nanoparticleassembly was centrifuged (3000 rpm for 2 min) in a polystyrene 1.5 mLvial, and the supernatant was removed. PBS buffer solution (700 μl) wasadded to the aggregate and the procedure was repeated to ensureisolation of the aggregate from unreacted protein and assay components.Then, 500 μl of water was added to the vial containing the aggregate todenature it. Microarrays were prepared and DNA hybridization methodswere used according to literature methods.^(11,22) The isolated DNAbiobarcodes were premixed with A2-modified nanoparticles or B2-modifiednanoparticles (2 nM), exposed to the DNA microarray, and incubated in ahybridization chamber (GRACE BIO-LABS) at room temperature for threehours. The array was then washed with 0.3M NaNO₃ and 10 nMNaH₂PO₄/Na₂HPO₄ buffer (pH 7) and submerged in Silver Enhancer Solution(Sigma) for three minutes at room temperature. The slide was washed withwater and then analyzed with a flat bed scanner.

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1. A method for detecting for the presence of a target analyte in asample comprising: providing a particle complex probe comprising aparticle having oligonucleotides bound thereto, a DNA barcode, and aoligonucleotide having bound thereto a specific binding complement to atarget analyte, wherein the DNA barcode has a sequence having at leasttwo portions, at least some of the oligonucleotides attached to theparticle have a sequence that is complementary to a first portion of aDNA barcode, the oligonucleotides having bound thereto a specificbinding complement have a sequence that is complementary to a secondportion of a DNA barcode, and wherein the DNA barcode is hybridized atleast to some of the oligonucleotides attached to the particle and tothe oligonucleotides having bound thereto the specific bindingcomplement; contacting the sample with a particle complex probe underconditions effective to allow specific binding interactions between theanalyte and the particle complex probe and to form an aggregated complexin the presence of analyte; and observing whether aggregate formationoccurred. 2-41. (canceled)